Composition for treating cancer and use thereof

ABSTRACT

The present invention provides a cyclic peptide containing RRXR motif. The present invention also provides a composition comprising the said cyclic peptide and a pharmaceutical acceptable carrier. The present invention further provides a method for treating cancer.

FIELD OF THE INVENTION

The present invention relates to a cyclic peptide containing RRXR motif.

The present invention also relates to a composition comprising the saidcyclic peptide and a pharmaceutical acceptable carrier.

The present invention further relates to a method for treating cancer.

BACKGROUND OF THE INVENTION

Neuropilin 1 (NRP1) was originally identified as a neuronal semaphorin3A receptor that mediates axonal extension during embryonic development.It was later discovered to be present in endothelial cells, mediatingangiogenesis during development and in lung cells, controlling lungbranching during development (Roche J, et al., Adv Exp Med Biol 2002,515:103-114). NRP1 is a type I transmembrane glycoprotein and acoreceptor for two extracellular ligands, semaphorins/collapsins, andvascular endothelial growth factor (VEGF; Ferrara N, et al., Nat Med2003, 9:669-976). VEGF mediates tumor angiogenesis and directly enhancestumor growth via VEGF/VEGF receptor (VEGFR) autocrine loops in tumors(Dias S, et al., Proc Natl Acad Sci USA 2001, 98:10857-10862). NRP1forms complexes with Flt-1 (VEGFR1) and Flk-1/KDR (VEGFR2) to enhancethe binding of VEGF₁₆₅ to VEGFRs and promotes VEGF₁₆₅-mediated tumorangiogenesis, cell migration, and tumorigenicity (Murga M, et al., Blood2005, 105:1992-1999).

NRP1 has been observed in cancer cells, including PC3 prostate cancercells and metastatic MDA-MB-231 breast cancer cells as well as severalother types of tumor cells (Lee M., Mol Cancer Ther 2006, 5:1099-1107).Overexpression of NRP1 enhances tumor angiogenesis and tumor growth invivo (Klagsbrun M, et al., Adv Exp Med Biol 2002, 515: 33-48). NRP1expression is present in various human cancers (Ellis LM. Mol CancerTher 2006, 5:1099-1107) and is associated with increased tumoraggressiveness and neovascularization; however, its modes of action arenot fully understood.

Lung cancer is the most common cause of cancer deaths, accounting for17% of deaths from cancer (Shibuya K, et al., BMC Cancer 2002, 2:37).Non-small cell lung carcinoma (NSCLC) is the predominant type of lungcancer (Hoffman P C, et al., Lancet 2000, 355:479-485). Metastasis isthe major cause of treatment failure and cancer deaths (Kwong Y L, etal., Chest 1997, 112: 1332-1337). The identification of metastasisenhancers and their signaling pathways may improve our understanding ofthe metastatic process and provide future targeted therapy for NSCLCpatients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows NRP1 is up-regulated in highly invasive human lungadenocarcinoma cell lines. A: close-up views of microarray imagesshowing NRP1 up-regulation in highly invasive CL1-5 and CL1-5-F4 cells.B: RT-PCR analysis of the differential expression of NRP1, semaphorin 3A(Sema3A), Flk-1/KDR, PLxA1, VEGF₁₆₅, and VEGF₁₂₁ in each of the CL1sublines. The GP-like gene was used as an internal control.

FIG. 2 shows Kaplan-Meier survival plots for patients with non-smallcell lung cancer grouped according to NRP1 mRNA expression. The relativeamount of tissue NRP1 mRNA, standardized against the amount of TATA-boxbinding protein mRNA, was expressed as −ΔC_(T)=[C_(T(NRP1))−C_(T(TBP))],where C_(T) is the threshold cycle. Patients were included in thehigh-expression group when the −ΔC_(T) was 0.32 (the median) or greater.A: the difference in disease-free survival between the high- andlow-expression patients was significant (P=0.0162). Tick marks: patientsfree of recurrence at their last follow-up. B: the difference in overallsurvival between the high- and low-expression patients was significant(P=0.0164). Tick marks: patients alive at their last follow-up. Allstatistical tests were two sided.

FIG. 3 shows suppression of NRP1 expression inhibited the invasion andmigration abilities of CL1-5 cells. A: expression of NRP1 shown byWestern blotting of tumor cells transfected by NRP1-siRNA-1,NRP1-siRNA-2, or nonsilencing siRNA. B: inhibition of CL1-5cell-invasive activity by NRP1 siRNAs. The invasive activity of cellswas detected by invasion assay. CL1-5 cells (2.5×10⁴) were seeded onTranswells coated with 30 μg Matrigel, transfected with NRP1-siRNA-1,NRP1-siRNA-2, or nonsilencing siRNA and incubated for 48 h. The cellsthat had invaded the membrane were then counted. Values were normalizedto the relative invasion activity of the reagent control. Experimentswere done in triplicate, three independent times. ANOVA revealed thatthe invasion ability was statistically significantly different among thecells treated with different concentrations of siRNAs, either siRNA-1 orsiRNA-2 (siRNA-1 group, P<0.001; siRNA-2 group, P=0.002). C: inhibitionof tumor cell migration by NRP1-siRNA determined by scratch woundhealing assay. CL1-5 cells were transfected with siRNA-1 for 24 h, andcells were scratched with a yellow pipette tip. The wound wasphotographed after the scratch, and cell numbers were counted within thegap area. Pictures of a representative assay at 0, 18, 21, and 24 h areshown here. D: NRP1-siRNA inhibition of tumor cell migration at varioustimes. The number of migrated cells within the gap area was countedafter the scratch. Columns: mean of four independent experiments; bars:SE.*indicates P<0.05, which is significantly different from nonsilencedcells. E: sNRP1 inhibition of tumor cell invasion ability as shown byinvasion assay. The relative invasion ability was statisticallysignificantly different among the cells treated with differentconcentrations of sNRP1 by ANOVA (P=0.002). F: sNRP1 inhibition ofF-actin polymerization and filopodia formation in CL1-5 cells. Red:F-actin.

FIG. 3 Continued. G: cells were infected with shLuc or shNRP1 lentivirusand then selected by culture medium containing 0.75 μg/mL puromycine for1 week. Left, Western blots of the cell lysates. Right, invasiveness ofthe treated cells. H: mice significantly developed more pulmonarymetastatic nodules after tail vein injection with CL1-5/shLuc cellscompared with CL1-5/shNRP-1cells. The lung metastatic nodules wererecorded and analyzed by the Student's t test (P=0.0063).

FIG. 4 shows that VEGF₁₆₅-induced NRP1 signaling involves VEGFR2phosphorylation, activation of PI3K and Akt phosphorylation. A: sNRP1inhibits phosphorylation of VEGFR2. Phosphorylation of VEGFR2 wasdetermined by immunoprecipitation with an anti-VEGFR2 antibody followedby Western blotting with an anti-phospho-VEGFR2 antibody. Total VEGFR2was determined by Western blot with an anti-VEGFR2 antibody. B: theeffect of NRP1-siRNA on PI3K activity. After siRNA-1 (NRP1) transfectionfor 48 h, CL1-5 cells were treated in RPMI-SF medium with 1.3nmol/LVEGF₁₆₅ for the indicated times. PI3K activity was detected asdescribed in Materials and Methods. C: inhibition of Akt phosphorylationby sNRP1 in CL1-5 cells. CL1-5 cells were treated with 1.3 nmol/LVEGF₁₆₅in the presence (a) or absence (b) of 10 nmol/L of sNRP1 for theindicated times. Akt and phosphorylated Akt proteins were detected byWestern blotting. The ratio of phosphorylated to total Akt isrepresented by Akt-p/Akt. D: the invasion ability was statisticallysignificantly different among the cells treated with differentconcentrations of wortmannin by ANOVA (P<0.001). E: the invasion abilitywas statistically significantly different among the cells treated withdifferent levels of LY294002 by ANOVA (P<0.001).

FIG. 5 shows cyclic 7-mer peptides bind NRP1 and inhibit CL1-5 invasionand angiogenesis in vivo. A: the selected peptides reducephosphorylation of VEGFR2. HUVECs were pretreated with peptides for 10min followed by treatment with VEGF for 5 min. Phosphorylation of VEGFR2was determined by Western blotting with an anti-phospho-VEGFR2 antibody.Total VEGFR2 was determined by Western blotting with an anti-VEGFR2antibody. B: the effect of peptides on CL1-5 cells invasive activity.The invasive activity of cells was detected by invasion assay. CL1-5cells (2.5×10⁴) were seeded on Transwells coated with 30 μg matrigel andincubated with peptides DG1 or DG2 for 48 h.

Then, the cells that had invaded the membrane were counted. Values werenormalized to the relative invasion activity of the nontreated controlcells. Experiments were done in triplicate, three independent times. InDG1- and DG2-treated cells, the invasion ability was statisticallysignificantly different across the various concentrations of DG1 or DG2by ANOVA (DG1, P<0.001; DG2, P=0.011). C: the effect of peptides ontumor angiogenesis in vivo. Immunohistochemical staining of the Matrigelplug sections with an anti-CD31 antibody showed a significant decreasein CD31-positive vessels in plugs containing DG1 peptide compared withmock-treated plugs. Original magnification, x200. The counts ofmicrovessels surrounding the tumor nests were calculated. D: effect ofpeptides on tumorigenesis in vivo. Volumes of tumors from control CL1-5cells (▴) and DG1-treated cells (▪) were measured at the indicated timesas described in Materials and Methods. Means and 95% CI are shown (n=5mice per group). E: summary diagram showing that VEGF₁₆₅ can bind toNRP1 and trigger the NRP1/VEGFR2/PI3K/Akt signaling pathways and resultin tumor angiogenesis, cancer cell invasion, and tumorigenesis. Thesynthetic peptides DG1/DG2 can specifically block this signaling pathwayand may have therapeutic potential.

SUMMARY OF THE INVENTION

The present invention provides a cyclic peptide containing RRXR motif.

The present invention also provides a composition comprising the saidcyclic peptide and a pharmaceutical acceptable carrier.

The present invention further provides a method for treating cancer.

DETAILED DESCRIPTION OF THE INVENTION

We identified by cDNA microarray that NRP1 expression is positivelycorrelated with the invasion ability of cancer cells in lung cancer cellline models (Chen J J, et al., Genomics 1998, 51:313-324). However, therole of NRP1 in cancer progression in NSCLC patients is not fullyunderstood. In the present invention, the role of NRP1 as an enhancerfor cancer invasion, metastasis, and angiogenesis and its signalingpathways, prognostic significance, and therapeutic implications iselucidated.

The present invention indicates that NRP1 is an enhancer of cancerinvasion and angiogenesis and is an independent predictor of cancerrelapse and poor survival in NSCLC patients. Suppression of NRP1signaling inhibits cancer invasion, tumorigenesis, angiogenesis, and invivo metastasis. The protumorigenic effect of NRP1 involves VEGF, PI3K,and Akt pathways. Two potent synthetic anti-NRP1 peptides (DG1 and DG2),which can block NRP1 signaling pathways, inhibit tumorigenesis, cancerinvasion, and angiogenesis, were identified. NRP1 is expected to be apotential biomarker for the selection of high-risk NSCLC patients foradjuvant chemotherapy, antiangiogenesis therapy, or other new targetedtherapies. This allows the maximization of potential therapeuticbenefits for high-risk patients and spare low-risk patients fromunnecessary treatment or toxicity. In the present invention, we showedthat NRP1 interacts with VEGFR2 to mediate VEGF-induced tumor invasion.VEGF mediates tumor angiogenesis and promotes migration and invasion oftumor cells by directly acting on its receptors via an endothelialcell-independent pathway. NRP1 alone has been shown to mediate breastcancer cell migration in a VEGFR2-independent manner, and in vitrostudies have shown that VEGFR1 activation by VEGF-A or VEGF-B incolorectal cancer cells leads to an increase in cell migration andinvasion. VEGF competes with semaphorin 3A for NRP1/plexin A1 complexbinding and enhances breast carcinoma migration via an autocrinepathway. NRP1 also inhibits migration, independent of semaphorin 3A, inpancreatic adenocarcinoma cells. DG1 and DG2 specifically inhibitedphosphorylation of VEGFR2 at Tyr¹²¹⁴ induced by VEGF165 in aconcentration-dependent manner. The role of NRP1 in enhancing tumorangiogenesis and tumor growth suggests that antagonizing NRP1 activityin tumor cells may be a feasible antitumor strategy. In the presentinvention, several small peptides with the consensus RRXR sequence motifspecifically block NRP1 signaling and suppress cancer cell invasion,tumorigenesis, and tumor angiogenesis. The minimal NRP1-bindingsynthetic peptide DG1 inhibited the invasive activity and in vivoangiogenesis of cancer cells without affecting cell viability. DG1 caninhibit VEGF₁₆₅-mediated downstream signaling and VEGFR2phosphorylation. Although no sequence homology was found between theselected peptides and VEGF₁₆₅, we found that peptides with positive netcharges and a cysteine-linked cyclic conformation are essential for NRP1binding.

NRP1 is a cancer invasion and angiogenesis enhancer. NRP1 is anindependent predictor of cancer relapse and poor survival in NSCLCpatients. NRP1 plays a critical role in tumorigenesis, cancer invasion,metastasis, and angiogenesis through VEGF, PI3K, and Akt pathways. NRP1is a potential new therapeutic target in NSCLC. Synthetic anti-NRP1peptides with conserved RRXR sequence motifs can block NRP1 signalingpathways and suppress tumorigenesis, cancer invasion, and angiogenesis.

Accordingly, the present invention provides a cyclic peptide containingRRXR motif, wherein R is arginine and X is any amino acid.

In one embodiment, the said cyclic peptide is DG1 of SEQ ID NO: 1 or DG2of SEQ ID NO: 2.

The present invention also provides a composition comprising the cyclicpeptide of claim 1 and a pharmaceutical acceptable carrier. In oneembodiment, the said cyclic peptide is DG1 or DG2.

The present invention further provides a method for treating cancer,comprising administering a subject with the above composition. Thecancer is breast cancer, colorectal cancer, esophageal cancer, gallbladder cancer, glioma, neuroblastoma, lung cancer, pancreatic cancer,prostate cancer. In one preferred embodiment, the cancer is lung cancer.In the most preferred embodiment, the cancer is non-small cell lungcancer.

In one embodiment, the subject is an animal. In more preferredembodiment, the subject is a mammal. In the most preferred embodiment,the subject is a human.

The cancer treatment of the present invention involves in inhibition ofcancer cell invasion, tumorigenesis, and tumor angiogenesis.

EXAMPLE

The examples below are non-limiting and are merely representative ofvarious aspects and features of the present invention.

Materials

Cells and Reagents.

Human lung cancer cell lines, CL1-0, CL1-1, CL1-5, and CL1-5-F4, wereestablished by selection of increasingly invasive cell populations froma clonal cell line of human lung adenocarcinoma, CL1 (Chu Y W, et al.,Am J Respir Cell Mol Biol 1997, 17:353-360). Human umbilical vascularendothelial cells (HUVEC) and culture media were purchased from CellApplications, Inc. Cell culture reagents were from Invitrogen. HumanVEGF165 was from PeproTech, Inc. Human anti-phospho-VEGFR2 antibody wasfrom Calbiochem. Anti-VEGFR2 (sc-504) antibody was from Santa CruzBiotechnology. Mouse antibody to phosphotyrosine (clone 4G10) was fromUpstate Biotechnology Inc. Anti-phospho-Akt, anti-Akt antibodies,wortmannin, and LY294002 were from New England Biolabs, and otherreagents were obtained from Sigma-Aldrich. Recombinant soluble NRP1(sNRP1) protein containing His6 and c-Myc domain tags was synthesized inNIH-3T3 murine fibroblast cells. Two cyclic peptides DG1 (CRRPRMLTC) SEQID NO: 1 and DG2 (CRSRRIRLC) SEQ ID NO: 2 were synthesized byDigitalgene (Taiwan). Primer sequences for reversetranscription-PCR(RT-PCR) and real-time PCR(r) analysis are shown inTable 2.

Patients and Tissue Specimens.

Sixty consecutive patients who underwent surgery for NSCLC at theNational Taiwan University Hospital from Sep. 1, 1994, to Apr. 30, 1998,were included in the study. This investigation was approved by theInstitutional Review Board of the National Taiwan University Hospital.None of the patients had received neoadjuvant chemotherapy or radiationtherapy before surgery. Specimens of lung cancer tissue obtained atsurgery were immediately snap-frozen in liquid nitrogen and stored at−80jC until use. The postsurgical pathologic stage of each tumor wasclassified according to the international tumor-node-metastasisclassification (Mountain C F. et al., Chest 1997, 111: 1710-1717). Thedemographic features of the patients are shown in Table 1.

TABLE 1 Clinicopathologic characteristics of 60 NSCLC patients Low NRP1High NRP1 expression expression patients (%), patients (%),Characteristic n = 30 n = 30 P Age (mean ± SD), y 64.4 ± 11.5 62.1 ±11.1 0.435* Sex Male 21 (70) 15 (50) 0.187^(†) Female  9 (30) 15 (50)Stage I and II 20 (67) 12 (40) 0.069^(†) III and IV 10 (33) 18 (60)Histology Adenocarcinoma 14 (47) 22 (73) 0.064^(†) Squamous 16 (53)  8(27) *t test. ^(†)Fisher's exact test.

TABLE 2 Primer and siRNA sequence and amplicon length Amplicon GeneForward primer Reverse primer length NRP1 GGCACACTCAGGGTCAAACTATGCCAACAGGCACAGTACA 455 (SEQ ID NO:3) (SEQ ID NO:4) Sema3AAGGAACTTGTCCC AGCAAAA ATGCAGCTCAGACACTCCTG 1190 (SEQ ID NO:5) (SEQ IDNO:6) VEGFR2 CTGGCATGGTCTTCTGTGAAGCA AATACCAGTGGATGTGATGCGG 793 (SEQ IDNO:7) (SEQ ID NO:8) PLxA1 AACCTGGAGAGCAAGAACCA GACTTGGTGAAG GTGGAGGA 602(SEQ ID NO:9) (SEQ ID NO:10) Gβ TACTGA TAACTTCTTGCTTCGTATGGAACCTGGCTAACTG 304 (SEQ ID NO:11) (SEQ ID NO:12) VEGFGTGAATGCAGACCAAAGAAAG AAACCCTGAGGGAGGCTC 96, 228 (SEQ ID NO:13) (SEQ IDNO:14) NRP1 CAGAAAAGCCCACGGTCAT CAGCCAAATTCACAGTTAAAACC 76 (r) (SEQ IDNO:15) (SEQ ID NO:16) TaqMan probe, (FAM)-ACAGCACCATACAATCAGAGTTTCCCACATA- (TAMRA). (SEQ ID NO:17) TBP(r) CACGAACCACGGCACTGATTTTTTCTTGCTGCCAGTCTGGAC 89 (SEQ ID NO:18) (SEQ ID NO:19) TaqMan probe(FAM)-TGTGCACAGGAGCCAAGAGTGAAGA-(TAMRA). (SEQ ID NO:20) *NonsilencingsiRNA AATTCTCCGAACGTGTCACGT (SEQ ID NO:21) *NRP1 siRNA#1AACACCTAGTGGAGTGATAAA (SEQ ID NO:22) *NRP1 siRNA#2 AACAGCCTTGAATGCACTTAT(SEQ ID NO:23) *Desalted small interfering RNA (siRNA) duplexes weresynthesized by Qiagen and were annealed following its standard protocol.siRNAs were transfected using the RNAiFect Transfection Reagent (Qiagen)according to the manufacturer's instructions.MethodsNRP1 mRNA Expression in Tumor Specimens from NSCLC Patients.

NRP1 expression in tumors from NSCLC patients was measured by real-timequantitative RT-PCR, based on TaqMan methodology, using the ABI PRISM7900 Sequence Detection System (Applied Biosystems; Heid C A, et al.,Genome Res 1994, 6: 986-994). The relative amounts of tissue NRP1 mRNAexpression were normalized with TATA-box binding protein mRNA andexpressed as −ΔCT=[CT_((NRP1))−CT_((TBP))]. Patients were included inthe high-expression group when −ACT was 0.32 (the median) or greater.The primer probe sets were designed and synthesized by AppliedBiosystems. The sequences of primers and small interfering RNAs (siRNA)used in this study are listed in Table 2. In Vitro invasion Assay.

A modified Boyden chamber system was used to investigate the invasivecapability of CL cells treated with selected peptides, sNRP1, and siRNAof NRP1 (Chu Y W, et al., Am J Respir Cell Mol Biol 1997, 17: 353-360).The polycarbonate membranes (containing 8-μm pores) of Transwell insertswere coated with Matrigel. The cells were suspended in RPMI 1640containing 10% NuSerum (Life Science), and 2.5×10⁴ cells were placedinto the upper well of each chamber. After incubation for 48 h at 37°C., the Transwell membrane was fixed with methanol for 10 min at roomtemperature and stained with a 50 μg/mL solution of propidium iodide(Sigma) for 30 min at room temperature. The number of cells in eachmembrane was counted under a microscope at a magnification of ×50 usingthe Analytical Imaging Station software package (Imaging Research Inc.).Each sample was assayed in triplicate.

Identification of NRP1-Binding Peptides by Phage Display.

A phage peptide library displaying cyclic random peptides (Ph.D.C. 7Cfrom New England Biolabs) was used for biopanning of NRP1. Recombinanthuman sNRP1 protein was coated onto the wells of polystyrene 96-wellplates and incubated with 2×10¹¹ plaque-forming units of the primarylibrary. Bound phages were eluted with glycine-HCl (pH, 2.2) andamplified in Escherichia coli (ER2738). Biopanning was repeated for fourrounds, with concentrations of Tween 20 in the wash solution increasingfrom 0.1% to 0.7%. Randomly selected phage clones from the fourth roundof panning were sequenced.

Surface Plasmon Resonance.

The binding kinetics of selected peptides with NRP1 were tested usingthe surface plasmon resonancebased measuring system (Biacore AB) at 25°C. Recombinant NRP1 was immobilized on CM5 sensor chips by aminecoupling at 400 response units using the amine coupling kit (Biacore)according to the manufacturer's instructions. Binding was detected inresonance units after injecting various concentrations of peptide at aflow rate of 30 μL/min. Sensograms of association and dissociation wererecorded and analyzed using BIAevaluation software 3.0 (Biacore AB).

VEGFR Tyrosine Phosphorylation.

VEGFR2 phosphorylation was assessed as previously described (Soker S, etal., J Cell Biochem 2002, 85:357-368). Briefly, CL1-5 cells were treatedwith a mixture of hVEGF and sNRP1 for 30 min on ice and then at 37° C.for 7 min. Cell lysates were immunoprecipitated with anti-VEGFR2antibodies. For Western blotting, the membranes were first probed withanti-Flk-1 antibodies and then reprobed with anti-phospho-VEGFR2antibodies ⅔(pc460) after being stripped with deblotting buffer. In theantiangiogenesis assay, HUVECs were pretreated with peptides for 10 minfollowed by treatment with VEGF for 5 min, and the cells were thenimmediately extracted with lysis buffer. Activation of Flk-1/KDR wasdetermined by immunoblotting cell extracts with anti-Flk-1 antibodiesand then reprobing with anti-phospho-VEGFR2 antibodies (pTyr¹²¹⁴) afterthe membranes had been stripped with deblotting buffer.

Phosphoinositide-3-Kinase Activity Assay.

Phosphoinositide-3-kinase (PI3K) activities were assayed as describedpreviously (Lin M T, et al., J Biol Chem 2001, 276: 48997-49002) withsome modifications. In brief, CL1-5 cell extracts were incubated withthe antiphosphotyrosine antibody and then precipitated with proteinA-Sepharose. The immunocomplexes were preincubated withphosphatidylinositol-4,5-P₂ (Sigma), and the kinase reaction wasinitiated by adding 10 μCi of [γ-³²P]ATP in reaction buffer for 15 min.Phospholipids were separated by TLC and visualized by phosphorimaging.

Wound Healing.

Cell migration was measured by the in vitro scratch wound healing assay(Tamura M, et al., Science 1998, 280: 1614-1617). CL1-5 cells weretransfected with 24 nmol/L siRNA-1 in 12-well plates. Twenty-four hoursafter transfection, cells were scratched with a yellow pipette tip andphotographed 18, 21, and 24 h after the scratch. The cell migration at0, 18, 21, and 24 h was evaluated by counting cells that had migratedfrom the wound edge.

Filamentous Actin Staining.

For filamentous actin (F-actin) staining, cells were seeded oncoverslips in 24-well plates and allowed to attach for 24 h in mediumcontaining 10% FCS.

The cells were fixed, washed, and permeabilized in 0.1% Triton-X. Thecells were incubated for 30 min with 5 units/mL of rhodamine-conjugatedphalloidin (Molecular Probe) and mounted using Fluor Save reagent(Calbiochem). The slides were analyzed using a Zeiss Axioplan 2microscope.

Experimental Metastasis In Vivo.

Cells were washed and resuspended in PBS. Subsequently, a single-cellsuspension containing 10⁶ cells in 0.1 mL of PBS was injected into thelateral tail veins of the 6-week-old severe combined immunodeficiency(SCID) mice (supplied by the animal center in the College of Medicine,National Taiwan University, Taipei, Taiwan). Mice were killed after 5weeks. The lungs were removed, weighed, and fixed in 10% formalin forfurther examination of metastasis formation. The number of lung tumorcolonies was counted under a dissecting microscope. All animalexperiments were done in accordance with the animal guidelines at theDepartment of Animal Care, Institute of Biomedical Sciences, AcademiaSinica, Taipei, Taiwan.

In Vivo Angiogenesis Assay.

All animal work was done under protocols approved by the InstitutionalAnimal Care and Use Committee of the College of Medicine, NationalTaiwan University. The effect of peptides on in vivo angiogenesis wasevaluated in the murine angiogenesis model using the Matrigel plug assayas described by Passaniti et al. (Passaniti A, et al., Lab Invest 1992,67: 519-528).

In Vivo Tumorigenesis Assay.

CL1-5 cells (2×10⁶) were mixed with or without peptides and thenimplanted into the flanks of the 6-weekold SCID mice. Injected mice wereexamined every 5 or 7 days for tumor appearance, and tumor volumes wereestimated from the length (a) and width (b) of the tumors, as measuredwith calipers, using the formula V=ab²/2. Mouse experiments wereapproved by the Laboratory Animal Center, Institute of BiomedicalSciences, Academia Sinica.

Statistical Analyses.

All data are presented as the means and 95% confidence intervals (95%CI) of at least three experiments. All statistical analyses were donewith the SAS Statistical Program (version 9.1; SAS Institute Inc.).Statistical significance was determined using an one-way ANOVA or asdescribed. Fisher's exact test was done to test associations betweencovariates and NRP1 for categorical data, and Student's t test was usedto test continuous variables. Survival curves were obtained by theKaplan-Meier method. Disease-free and overall survival of patients withlow versus high expression of NRP1 was analyzed using the log-rank test.Multivariate Cox proportional-hazards regression was done with overallor disease-free survival as the response variable. P<0.05 was consideredstatistically significant.

Example 1 NRP1 Expression Correlates with the Invasive Ability of LungCancer Cells

Five distinct lung tumor cell lines with progressive invasiveness wereestablished previously. Microarray analysis showed that NRP1 wasup-regulated in the highly invasive NSCLC cell lines, CL1-5 and CL1-5-F4(FIG. 1A). NRP1 and its coreceptor VEGFR2 were only expressed in thehighly invasive CL1-5 and CL1-5-F4 cells. Expression of the NRP1 ligandsemaphorin 3A was down-regulated in an opposite pattern to NRP1. Therewere no differences in VEGF or plexin A1 expression in this cell panel(FIG. 1B).

Example 2 NRP1 mRNA Expression Correlates with Cancer Relapse andSurvival in NSCLC Patients

Real-time quantitative RT-PCR was used to determine the number of NRP1transcripts in lung cancer tissues from 60 patients with NSCLC. Wearbitrarily used the median value to classify patients into high- orlow-expression groups. The clinicopathologic characteristics of the 60NSCLC patients are shown in Table 1. Patients with high NRP1 expressionhad shorter disease-free (P=0.0162) and overall survival (P=0.0164)compared with low NRP1-expression patients (FIG. 2). Multivariate Cox'sproportional hazards regression analyses showed that low NRP1 expressionwas associated with overall survival of NSCLC patients independent ofclinicopathologic stage, age, sex, and cell type [0 for low and 1 forhigh NRP1 expression, respectively; hazard ratio (HR), 2.37; 95% CI,1.15-4.9; P=0.0196]. Similarly, the hazard ratio for disease-freesurvival remained significant only for the expression of NRP1 (HR, 2.38;95% CI, 1.15-4.91; P=0.0195).

Example 3 Endogenous NRP1 Expression Knockdown Suppresses Cancer CellInvasion

To knock down NRP1 expression, two individual siRNAs directed againstthe NRP1 gene were transfected into NRP1-positive lung cancer cellsCL1-5. Significant suppression of NRP1 expression was achieved bysiRNA-1 and siRNA-2 (FIG. 3A). Both NRP1 siRNAs decreased the invasionability of CL1-5 cells in a dose-dependent manner compared with thenonsilencing siRNA control (FIG. 3B). To examine whether theanti-invasion activity of the NRP1-specific siRNAs is associated withsuppression of cell mobility, the effects of NRP1-specific siRNA1 on themigration capability of cells were analyzed. CL1-5 cells weretransfected with siRNA-1 or nonsilencing control siRNA; the migrationability was determined by the scratch wound healing assay. That NRP1siRNAs can suppress CL1-5 cell mobility, and migration capability wasshown by the scratch wound healing assay (FIG. 3C). NRP1-siRNAssignificantly inhibited the migration of CL1-5 cells at 24 h (FIG. 3D).

Example 4 Soluble NRP1 Inhibits Cancer Cell Invasion and FilopodiaFormation

Recombinant sNRP1 was expressed in human fibroblast (NIH-3T3) cells andwas secreted into cultured medium sNRP1 proteins were purified from theconditioned medium by ammonium sulfate precipitation and then by Ni-NTAcolumn purification on a fast protein liquid chromatography (FPLC)system. The binding affinity of the recombinant sNRP1 to VEGF₁₆₅ wasdetermined by surface plasmon resonance analysis. The averagedissociation constant (KD) of the human VEGF₁₆₅ binding to sNRP1 was 125nmol/L, consistent with previous results obtained using the sametechnology (Dias S, et al., Proc Natl Acad Sci USA 2001, 98:10857-10862). sNRP1 was expressed differently from intact NRP1 andseemed to be a VEGF₁₆₅ antagonist. There was a dose-dependent decreasein the invasion ability of CL1-5 cells after treatment with sNRP1 (FIG.3E). The F-actin of CL1-5 cells was stained with rhodamineconjugatedphalloidin and examined by fluorescence microscopy. sNRP1 inhibitedF-actin polymerization and filopodia formation in CL1-5 cells in adose-dependent manner (FIG. 3F).

Example 5 Knockdown of Endogenous NRP1 Expression Suppresses CancerMetastasis In Vivo

Knockdown of endogenous NRP1 expression in CL1-5 cells by shRNAlentivirus significantly reduced the invasive activity by 50% (FIG. 3G).Mice injected with CL1-5/shNRP1 cells developed significantly fewerpulmonary metastatic nodules than those with CL1-5/shLuc cells (FIG.3H).

Example 6 NRP1 Signaling Pathways Involve VEGFR2, PI3K, and AktActivation

To identify the signaling pathways affected by NRP1, CL1-5 cells weretreated with VEGF₁₆₅ for various periods and analyzed signalingintermediates. CL1-5 cells were treated with VEGF₁₆₅ and sNRP1, and thephosphorylation of VEGFR2 was determined by immunoprecipitation with ananti-VEGFR2 antibody followed by Western blotting with ananti-phospho-VEGFR2 antibody. VEGF₁₆₅-induced VEGFR2 activation wasdecreased by sNRP1 in a dose-dependent manner and was totally blocked byhigh concentrations of sNRP1 (FIG. 4A). VEGF₁₆₅ induced PI3K activationwith peak phosphorylation at 30 min, which returned to the baselinelevels by 60 min. The addition of siRNA-1 decreased VEGF₁₆₅-induced PI3Kactivation in CL1-5 cells compared with the nonsilencing siRNA-1 control(FIG. 4B). Phosphorylation of Akt, a downstream mediator of PI3K, isinvolved in NRP1 modulation of VEGF actions. VEGF₁₆₅-inducedphosphorylation of Akt at Ser473 in CL1-5 cells was decreased to lessthan one third in the presence of sNRP1 (FIG. 4C). Both PI3K inhibitors,wortmannin and LY294002, decreased the invasion ability of CL1-5 cells(ANOVA: wortmannin, P<0.001; LY294002, P<0.001; FIGS. 4D and E).

Example 7 RRXR-Containing Peptides can Inhibit NRP1-Mediated VEGFR2Phosphorylation

To identify whether any new signature motif can bind and inhibitNRP1-mediated invasion, mammalian cell-expressed NRP1 proteins were usedas bait to screen a random cyclic 7-mer peptide library for NRP1-bindingpeptides. A Ph.D. C7C phage display library containing 10¹¹ randomcyclic 7-amino acid peptides was applied for biopanning. After fourrounds of screening, 63 clones were isolated. DNA sequencing showed thatalmost all selected peptides contained arginine (R) residues. Aconsensus motif, -RRXR-, was found in nine clones by MULTALIN programalignment (Table 3). The two most potent peptides (cyclic 9-merpeptides, DG1, and DG2) were selected and chemically synthesized forfurther analysis of their binding kinetics and NRP1 inhibition. Surfaceplasmon resonance was used to measure the real-time association anddissociation of the binding of RRXR-containing peptides to NRP1. Theaverage dissociation constants (K_(D)) for the binding of DG1 and DG2 toNRP1 were 1.40±0.23 and 5.37±0.49 μmol/L, respectively (Table 4). Theslightly higher binding affinity of DG1 to NRP1 was due to a morefavorable k_(a). No binding was observed to either the immobilizedVEGFR1 or VEGFR2 sensor chips (data not shown). DG1 and DG2 specificallyinhibited VEGF₁₆₅-induced phosphorylation of VEGFR2 at Tyr¹²¹⁴ in aconcentration-dependent manner with a significant effect at 40 μmol/Land almost complete inhibition at 120 μmol/L (FIG. 5A).

TABLE 3 The RRXR motif of the peptides selected by binding to NRP1Peptide number Sequence 4-1     R R P R M L T 4-2 Q L R R Q R R 4-3 H SR R M R K 4-5 R S R R I R L 4-9 M K R R P R K 4-28     R R L R R R R4-40 P I R R Q R L 4-43     R R S R Q S R 4-53 H K R R I R Q Consensus  - R R X R -

TABLE 4 Kinetic constants for the interaction of the selected peptideswith NRP1 Peptide K_(D), μmol/L k_(a,) (mol/L)⁻¹ s⁻¹ k_(d) × 10⁻⁴, s⁻¹DG1 1.40 ± 0.23 1,229 ± 246 17.2 ± 2.25 DG2 5.37 ± 0.49 123.4 ± 24  6.63± 0.81 NOTE: The kinetic constants were determined using the Blacoresystem as described in Materials and Methods.

Example 8 RRXR-Containing Peptides Inhibit Cancer Cell Invasion,Tumorigenesis, and Tumor Angiogenesis

The in vitro invasion assay was done using the highly invasive CL1-5cells to investigate the effects of DG1 and DG2 on the invasiveness ofthe lung carcinoma cells. Treatment with DG1 or DG2 peptides inhibitedCL1-5 cell invasion in a dose-dependent manner (FIG. 5B). DG1 reducedthe number of cancer cells invading through the Matrigel by 70%, and DG2reduced the number of cancer cells by 50%. This suggests that theinteraction of the peptides with NRP1 may be associated withNRP1-mediated cancer cell invasion. The treatment was not cytotoxic,suggesting that the decreased number of invading cells was due to theinhibitory effect of the RRXR-containing peptides on the invasivephenotype. To understand whether DG1 can reduce angiogenesis ortumorigenesis, in vivo angiogenesis and xenograft tumor assays weredone. DG1 inhibited tumor angiogenesis in vivo (FIG. 5C). The tumormicrovascular count from DG1-treated CL1-5 cells (75±4; in ×200 fields)was significantly less than that of the untreated tumor cells (227±33;in ×200 fields). The tumor angiogenesis activity of DG1-treated CL1-5cells decreased significantly by 3-fold compared with the untreatedtumor cells. The effect of DG1 on tumorigenicity in vivo was testedusing the xenograft tumor assay. DG-1 treatment reduced tumor volume to60.1 mm3 (95% CI, 27.6-92.6 mm³) in mice 21 days after the inoculationof the CL1-5 cells, compared with the tumor volume of 464.1 mm³ (95% CI,200.1-728.2 mm3; P=0.003) without DG-1 treatment (FIG. 5D).

While the invention has been described and exemplified in sufficientdetail for those skilled in this art to make and use it, variousalternatives, modifications, and improvements should be apparent withoutdeparting from the spirit and scope of the invention.

1. A synthetic anti-NRP1 cyclic peptide for blocking NRP1 signalingpathways, consisting of SEQ ID NO: 1 or SEQ ID NO:
 2. 2. A compositioncomprising the cyclic peptide of claim 1 and a pharmaceutical acceptablecarrier.